Retraction and Purification of gbr22 from E. coli Cultures
Introduction
The bacteria Escherichia coli have been developed to produce proteins by over-expressing the protein of interest. In the bacteria, the proteins in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc and it makes it possible to study human proteins without having to use human tissues. At the same time, non-native proteins are produced because techniques such as PCR allow DNA to be manipulated. pGEM-gbr22 is a plasmid that encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef and its purple color allows its expression and purification to be easily followed. The protein in this lab, gbr22 has six histidine residues attached at the C-terminus, which is used to distinguish this protein from other cellular proteins. Other important parts include the hexa-histidine tag, an affinity tag that allows for fast and efficient purification of the protein, and the expression plasmid inserted into the host E.coli BL21, which carries a gene for ampicillin resistance.
The histitine residues bind to divalent cations and is immobilized on a column matrix such as Ni-NTA agarose. Ni-NTA affinity chromatography, a method of protein purification, is utilized by separating the molecules after the bacteria host is lysed. Step by step, waste is washed away. Because imidazole competes with histidine residues for metal binding, its presence releases protein. All these are achieved due to the affinity of the 6xHis-tagged protein for divalent ions. So, elution solutions are produced when imidazole is added and finally, the protein is purified.
The step of protein purification needs to be verified and one of the ways is through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the most commonly used technique. This monitors the success of each separation step. Spectroscopy can measure the concentration of proteins in solution, but fails to fully distinguish the different proteins in a mixture. In gel electrophoresis, the samples are introduced at one end of a porous gel and an electrical field is applied across the gel. It uses SDS, an anionic detergent which denatures proteins and produces a negative charge that is proportional to its mass, allowing the distance of migration to be related to the mass of the protein. From this, the mass can be estimated by comparison to molecular weight standards. In this lab, competent E. coli cells were transformed. Through this, the protein gbr22 was retracted and purified. Finally, electrophoresis was performed to validate the process of purification.
Materials and Methods
This lab was divided into three parts: expression, purification, and characterization. To express the recombinant protein, the materials needed included an ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear, sterile round-bottom tubes, 37oC incubator, colirollers, 2 LB Agar Amp plates, 1 spare Agar plate w/o antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette and tips. Through this lab, competent bacterial cells with a DNA plasmid was transformed, a starter culture of bacteria was grown, and this overnight culture was used to inoculate a larger culture and express the recombinant protein. Sample 1 of lysed cells was collected. At the end, the cells were harvested and frozen for purification.
To purify the protein, the materials needed were a beaker of water, 1M Imidazole (10x PBS and 1x PBS), 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x15 ml conical tubes, bio-rad Econo chromatography column, ring stand and clamps, Ni-NTA resin, and benzonase. Bacterial cells were lysed to release the soluble proteins (sample 2). Then, insoluble cell debris (cell wall, membranes, inclusion bodies) was removed by centrifugation (sample 3) and the protein was purified using the affinity tag and Ni-NTA resin (samples 4 - 6). At the end, six samples were collected.
For protein characterization, the previous protein samples were analyzed. The materials were mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, bio-Rad precast polyacrylamide gel, 100 ml 6x gel loading buffer, protein samples, MW standards, plastic container w/ lid, Imperial protein stain. SDS-PAGE was used to separate proteins in the samples. By using electrophoresis and spectroscopy, the molecular weight of the gbr22 protein as well as the purity and yield of the final product were estimated. This data was correlated to the previous UV-Vis spectroscopy measurements to estimate the concentration of the protein solution.
Results
Figure 1: Ampicillin plate with plasmid DNA; after one day of cultivation; bacterial growth
Figure 2: Control Ampicillin plate with no DNA added; Fun plate (on the right)
Figure 3: LB Broth with Purple Culture after incubation (Day 3)
Figure 4: Cell pelle with purple colony after centrifugation
Figure 5: Elution 1 and Elution 2 from Protein Purification lab
Figure 6: Nanodrop spectophotometry Trial 1
Figure 7: Nanodrop spectophotometry Trial 2
Figure 8: UV/Vis spectophotometry Trial 1
Figure 9: UV/Vis spectophotometry Trial 2
Figure 10: Protein gel with ladder & 6 samples
Figure 11: Protein gel after unstaining
Figure 12: Fermentas pageruler pre-stained protein ladder
From the transformation plate image, there were hundreds of colonies that were spread out in the petri dish. After the sample was centrifuged, the pellet produced was weighed to be 0.38 g. For the Nanodrop spectrophotometry calculation, the yield at a 280 wavelength was 1.5976 x 10^(-4) mg and the yield at max wavelength was 9.2654 x 10^(-5) mg. For electrophoresis, the marker used was Fermentas pageruler pre-stained protein ladder.
Absorbance at 280 = 0.064 x 10 = 0.64 Absorbance at 574 = 0.113 x 10 = 1.13 Extinction coefficient at 280 nm = 38,850 Extinction coefficient at max = 118,300 MW of gbr22 = 25794.2 A=εbc "280" 0.64= (38,850) (1)C C=1.647 x 10^(-5) "Max" 1.13= (118,300) (1)C C=9.552 x 10^(-6) Yield at 280 nm: 9.7(1.647 x 10^(-5)) = 1.5976 x 10^(-4) mg Yield at max nm: 9,7 (9.552 x 10^(-6)) = 9.2654 x 10^(-5) mg
Discussion
From the expression part of the lab, sample 1 was collected. This was the cell lysate and the expressed protein. Sample 2 was the supernatant of sample 1 after it was centrifuged. This means that sample 2 should be more concentrated than sample 1 because everything has been separated. Sample 3 was more washed out when the resin and buffer were added to wash out the "waste" and sample 4 was a "wash" by weak version of imidazole. For samples 5 and 6, gbr22 protein was finally released from the Ni-NTA resin by using a buffer with a high concentration of imidazole. For elution one and elution 2, all the other materials were washed out and finally, only the protein was contained in elution 2.
Because of this, samples 3 and 4 were not as concentrated as samples 1 and 2. Sample 5 had a strong band around 100 kDa and some other weak bands. This is because by this point, most of the other materials have been washed out. In sample 6, this strong band basically became the only band and it was no longer as strong. This is the end of protein purification because this was the only band remaining, showing that only the interest protein was left. The molecular weight of gbr22 was estimated to be about 100 kDa and the purity level should be very close to 100% because no other band had the intensity close to the intensity of the protein band.
Several errors that could have evolved included different sources of contaminations. Also, during the purification process, parts of the protein or some of the waste could have been left behind. Finally, during electrophoresis, the tape was forgotten to be taken off, so some issues with running the gel arose. This could have led to the underdevelopment of the running of the gel, which would change the molecular weight of the protein.
Conclusion
In this lab, the protein gbr22 was expressed from a culture of E. coli bacteria, purified through chromatography and spectrophotometry, and verified by gel electrophoresis. This technique was able to separate the protein of interest from other materials and now the protein can be studied separately. Throughout this lab, different important techniques were practiced that could all be used in the future - the growth of a colony, the purification of protein, and gel electrophoresis.
Retraction and Purification of gbr22 from E. coli Cultures
Introduction
The bacteria Escherichia coli have been developed to produce proteins by over-expressing the protein of interest. In the bacteria, the proteins in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc and it makes it possible to study human proteins without having to use human tissues. At the same time, non-native proteins are produced because techniques such as PCR allow DNA to be manipulated. pGEM-gbr22 is a plasmid that encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef and its purple color allows its expression and purification to be easily followed. The protein in this lab, gbr22 has six histidine residues attached at the C-terminus, which is used to distinguish this protein from other cellular proteins. Other important parts include the hexa-histidine tag, an affinity tag that allows for fast and efficient purification of the protein, and the expression plasmid inserted into the host E.coli BL21, which carries a gene for ampicillin resistance.
The histitine residues bind to divalent cations and is immobilized on a column matrix such as Ni-NTA agarose. Ni-NTA affinity chromatography, a method of protein purification, is utilized by separating the molecules after the bacteria host is lysed. Step by step, waste is washed away. Because imidazole competes with histidine residues for metal binding, its presence releases protein. All these are achieved due to the affinity of the 6xHis-tagged protein for divalent ions. So, elution solutions are produced when imidazole is added and finally, the protein is purified.
The step of protein purification needs to be verified and one of the ways is through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the most commonly used technique. This monitors the success of each separation step. Spectroscopy can measure the concentration of proteins in solution, but fails to fully distinguish the different proteins in a mixture. In gel electrophoresis, the samples are introduced at one end of a porous gel and an electrical field is applied across the gel. It uses SDS, an anionic detergent which denatures proteins and produces a negative charge that is proportional to its mass, allowing the distance of migration to be related to the mass of the protein. From this, the mass can be estimated by comparison to molecular weight standards. In this lab, competent E. coli cells were transformed. Through this, the protein gbr22 was retracted and purified. Finally, electrophoresis was performed to validate the process of purification.
Materials and Methods
This lab was divided into three parts: expression, purification, and characterization. To express the recombinant protein, the materials needed included an ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear, sterile round-bottom tubes, 37oC incubator, colirollers, 2 LB Agar Amp plates, 1 spare Agar plate w/o antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette and tips. Through this lab, competent bacterial cells with a DNA plasmid was transformed, a starter culture of bacteria was grown, and this overnight culture was used to inoculate a larger culture and express the recombinant protein. Sample 1 of lysed cells was collected. At the end, the cells were harvested and frozen for purification.
To purify the protein, the materials needed were a beaker of water, 1M Imidazole (10x PBS and 1x PBS), 1.7 ml centrifuge tubes, 2 x 10 ml round bottom tubes, 4 x15 ml conical tubes, bio-rad Econo chromatography column, ring stand and clamps, Ni-NTA resin, and benzonase. Bacterial cells were lysed to release the soluble proteins (sample 2). Then, insoluble cell debris (cell wall, membranes, inclusion bodies) was removed by centrifugation (sample 3) and the protein was purified using the affinity tag and Ni-NTA resin (samples 4 - 6). At the end, six samples were collected.
For protein characterization, the previous protein samples were analyzed. The materials were mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, bio-Rad precast polyacrylamide gel, 100 ml 6x gel loading buffer, protein samples, MW standards, plastic container w/ lid, Imperial protein stain. SDS-PAGE was used to separate proteins in the samples. By using electrophoresis and spectroscopy, the molecular weight of the gbr22 protein as well as the purity and yield of the final product were estimated. This data was correlated to the previous UV-Vis spectroscopy measurements to estimate the concentration of the protein solution.
Results
From the transformation plate image, there were hundreds of colonies that were spread out in the petri dish. After the sample was centrifuged, the pellet produced was weighed to be 0.38 g. For the Nanodrop spectrophotometry calculation, the yield at a 280 wavelength was 1.5976 x 10^(-4) mg and the yield at max wavelength was 9.2654 x 10^(-5) mg. For electrophoresis, the marker used was Fermentas pageruler pre-stained protein ladder.
Absorbance at 280 = 0.064 x 10 = 0.64
Absorbance at 574 = 0.113 x 10 = 1.13
Extinction coefficient at 280 nm = 38,850
Extinction coefficient at max = 118,300
MW of gbr22 = 25794.2
A=εbc
"280" 0.64= (38,850) (1)C
C=1.647 x 10^(-5)
"Max" 1.13= (118,300) (1)C
C=9.552 x 10^(-6)
Yield at 280 nm: 9.7(1.647 x 10^(-5)) = 1.5976 x 10^(-4) mg
Yield at max nm: 9,7 (9.552 x 10^(-6)) = 9.2654 x 10^(-5) mg
Discussion
From the expression part of the lab, sample 1 was collected. This was the cell lysate and the expressed protein. Sample 2 was the supernatant of sample 1 after it was centrifuged. This means that sample 2 should be more concentrated than sample 1 because everything has been separated. Sample 3 was more washed out when the resin and buffer were added to wash out the "waste" and sample 4 was a "wash" by weak version of imidazole. For samples 5 and 6, gbr22 protein was finally released from the Ni-NTA resin by using a buffer with a high concentration of imidazole. For elution one and elution 2, all the other materials were washed out and finally, only the protein was contained in elution 2.
Because of this, samples 3 and 4 were not as concentrated as samples 1 and 2. Sample 5 had a strong band around 100 kDa and some other weak bands. This is because by this point, most of the other materials have been washed out. In sample 6, this strong band basically became the only band and it was no longer as strong. This is the end of protein purification because this was the only band remaining, showing that only the interest protein was left. The molecular weight of gbr22 was estimated to be about 100 kDa and the purity level should be very close to 100% because no other band had the intensity close to the intensity of the protein band.
Several errors that could have evolved included different sources of contaminations. Also, during the purification process, parts of the protein or some of the waste could have been left behind. Finally, during electrophoresis, the tape was forgotten to be taken off, so some issues with running the gel arose. This could have led to the underdevelopment of the running of the gel, which would change the molecular weight of the protein.
Conclusion
In this lab, the protein gbr22 was expressed from a culture of E. coli bacteria, purified through chromatography and spectrophotometry, and verified by gel electrophoresis. This technique was able to separate the protein of interest from other materials and now the protein can be studied separately. Throughout this lab, different important techniques were practiced that could all be used in the future - the growth of a colony, the purification of protein, and gel electrophoresis.
References
Affinity Chromatography. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html (accessed April 17, 2011).
Fermentas Molecular Biological Tools. PageRuler Prestained Protein Ladder. http://www.fermentas.com/en/products/all/protein-electrophoresis/prestained-ladders/sm067-pageruler- (accessed April 17, 2011).